Citric acid production from xylan and xylan hydrolysate by semi-solid culture of Aspergillus niger

Citric acid production from xylan and xylan hydrolysate was done by Aspergillus niger Yang no. 2 cultivated in a semi-solid culture using bagasse as a carrier. Yang no. 2 produced 72.4 g/l and 52.6 g/l of citric acid in 5 d from 140 g/l of xylose and arabinose, respectively. Yang no. 2 produced 51.6 g/l of citric acid in 3 d from a concentrated xylan hydrolysate prepared by cellulase treatment, containing 100 g/l of reducing sugars. Moreover, Yang no. 2 directly produced 39.6 g/l of citric acid maximally in 3 d from 140 g/l of xylan.     

Screening for distinct xylan degrading enzymes in complex shake flask fermentation supernatants

The efficient degradation of complex xylans needs collaboration of many xylan degrading enzymes. Assays for xylan degrading activities based on reducing sugars or PNP substrates are not indicative for the presence of enzymes able to degrade complex xylans: They do not provide insight into the possible presence of xylanase-accessory enzymes within enzyme mixtures. A new screening method is described, by which specific xylan modifying enzymes can be detected.Fermentation supernatants of 78 different fungal soil isolates grown on wheat straw were analyzed by HPLC and MS. This strategy is powerful in recognizing xylanases, arabinoxylan hydrolases, acetyl xylan esterases and glucuronidases.No fungus produced all enzymes necessary to totally degrade the substrates tested. Some fungi produce high levels of xylanase active against linear xylan, but are unable to degrade complex xylans. Other fungi producing relative low levels of xylanase secrete many useful accessory enzyme component(s).     

Effect of Carbon Source on Production of Lytic Enzymes by the Sclerotial Parasites Trichoderma atroviride and Coniothyrium minitans

Three isolates of Trichoderma atroviride and two isolates of Coniothyrium minitans known to efficiently degrade sclerotia of Sclerotinia sclerotiorum were cultured on minimal medium with sucrose, carboxymethyl cellulose (CMC), xylan, laminarin, colloidal chitin or powdered sclerotia as carbon source. The activity of endochitinase, endo‐β‐1,3‐glucanase, endoxylanase and endocellulase in culture filtrates was determined after 7 and 15 days of culture using dye‐labelled substrates. The strongest inducers of chitinase were colloidal chitin and sclerotia powder. Chitinase activity appeared to be faster induced in the isolates of T. atroviride than in the isolates of C. minitans, but the maximum level of activity present in culture filtrates of the two species was similar. With CMC and xylan as carbon source, concurrent production of the corresponding enzymes was observed for the Trichoderma isolates. The isolates of C. minitans produced cellulase on xylan but not on CMC, whereas xylanase was produced on both carbon sources. Laminarin induced the formation of glucanases in the three isolates of T. atroviride but not the isolates of C. minitans. However, in the sclerotia‐containing cultures of C. minitans glucanase activity was even higher than in the respective cultures of the Trichoderma isolates. During the 31‐day study period, the pattern of enzyme production in shake cultures containing sclerotia powder was very similar for the isolates of T. atroviride and C. minitans. Glucanase activity generally reached a peak 24 days after inoculation of the flasks, whereas the activity of chitinase, cellulase and xylanase remained fairly constant throughout the experiment.     

Enzymatic synthesis of alkyl xylobioside and xyloside from xylan and alcohol

Summary Alkyl -D-xylobioside and alkyl -D-xyloside were prepared by the one-pot reaction of xylan and a fatty alcohol, such as 1-octanol, 1-decanol, 2-octanol and 2-ethylhexanol using the cell-free culture filtrate of the xylan-assimilating strain, Aureobasidium pullulans KK415. Using this strain, a novel surfactant, alkyl -D-xylobioside, was produced as the main product when the alcohol and xylan was incubated at a temperature of 65 °C and pH 4.5.     

One-pot synthesis of alkyl β-D-xylobioside from xylan and alcohol by acetone powder of Aureobasidium pullulans

Alkyl β-D-xylobioside was prepared by the one-pot reaction of xylan and a fatty alcohol using acetone powder (acetone-dried cells) of Aureobasidium pullulans KK415 (ATCC 201145). Both the yields and reaction speed were significantly increased using the acetone powder compared to those of the cell-free culture filtrate. Using 0.066% acetone powder, 2-ethylhexyl, 1-octyl and 2-octyl b-D-xylobiosides were produced in the yields of 118, 80 and 74 mg per g xylan, respectively, when alcohol and xylan were incubated at 65°C for 16 h. On the other hand, C8 alkyl xylo-bioside was hardly produced by the resting cells of A. pullulans.     

Hyperthermostable acetyl xylan esterase

An esterase which is encoded within a Thermotoga maritima chromosomal gene cluster for xylan degradation and utilization was characterized after heterologous expression of the corresponding gene in Escherichia coli and purification of the enzyme. The enzyme, designated AxeA, shares amino acid sequence similarity and its broad substrate specificity with the acetyl xylan esterase from Bacillus pumilus, the cephalosporin C deacetylase from Bacillus subtilis, and other (putative) esterases, allowing its classification as a member of carbohydrate esterase family 7. The recombinant enzyme displayed activity with p-nitrophenyl-acetate as well as with various acetylated sugar substrates such as glucose penta-acetate, acetylated oat spelts xylan and DMSO (dimethyl sulfoxide)-extracted beechwood xylan, and with cephalosporin C. Thermotoga maritima AxeA represents the most thermostable acetyl xylan esterase known to date. In a 10 min assay at its optimum pH of 6.5 the enzyme's activity peaked at 90°C. The inactivation half-life of AxeA at a protein concentration of 0.3 µg µl⁻¹ in the absence of substrate was about 13 h at 98°C and about 67 h at 90°C. Differential scanning calorimetry analysis of the thermal stability of AxeA corroborated its extreme heat resistance. A multi-phasic unfolding behaviour was found, with two apparent exothermic peaks at approximately 100–104°C and 107.5°C. In accordance with the crystal structure, gel filtration analysis at ambient temperature revealed that the enzyme has as a homohexameric oligomerization state, but a dimeric form was also found.     

Green synthesis of xylan hemicellulose esters

The esterification of xylan type hemicelluloses, isolated from birchwood, was carried out firstly in homogeneous conditions using N,N-dimethylformamide (DMF) and lithium chloride (LiCl) in the presence of 4-dimethylaminipyridine (DMAP). The degree of substitution (DS) of xylan acetates ranged between 0.9 and 2.0 as a function of experimental conditions. Due to the problems of toxicity and recycling of DMF, an alternative method of esterification is reported in the second part of this work, performing in the absence of organic solvent and using DMAP or methanesulfonic acid (MSA) as catalysts. Acetylation reaction catalyzed by MSA was developed through an experimental design in order to achieve the highest DS under the mildest conditions. The significant factors and their interactions were identified. The optimization of reaction parameters allowed to obtain a high DS (1.6) and maximal yield (85%). Moreover, the reactivity of propionic and hexanoic anhydrides was evaluated and hydrophobic xylan esters with low degrees of substitution were obtained.     

Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose

Cell wall hemicelluloses and pectins are O‐acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O‐acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody‐tissue‐specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall‐bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a β‐1,4‐endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ~70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1‐expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production.     

Saccharification of xylan by an amyloglucosidase of Termitomyces clypeatus and synergism in the presence of xylanase

Summary An amyloglucosidase from a mycelial culture of the mushroom Termitomyces clypeatus hydrolysed larch wood xylan independently and synergistically with an endo-(14) xylanase of the same fungus. The glucoamylase saccharified xylan predigested with xylanase at a faster rate compared to that of xylanase acting on amylase-digested xylan. However, overall saccharification of xylan in both cases was the same. Only glucose was liberated from xylan by amylase digestion whereas xylose, xylobiose and other oligosaccharides were liberated during xylanase digestion. The synergistic response of enzyme combinations was reflected in the liberation of glucose from xylan, rather than xylose. Glucoamylase and xylanase activities on soluble and insoluble fractions of larch wood xylan with different xylose and glucose contents suggested that synergism in xylanolysis by the presence of glucoamylase was dependent on the activity of the participating xylanase on the xylan preparation. It is suggested that possibly -glucosidic linkages are present in xylan and that amyloglucosidase might be involved in xylanolysis. Correspondence to: S. Sengupta     

Characterization of several bovine rumen bacteria isolated with a xylan medium

Dehority, B. A. (Ohio Agricultural Research and Development Center, Wooster). Characterization of several bovine rumen bacteria isolated with a xylan medium. J. Bacteriol. 91:1724-1729. 1966.-Studies were conducted to characterize eight strains of bacteria isolated from bovine rumen contents, by use of a medium containing xylan as the only added carbohydrate source. Based on morphology, biochemical reactions, nutritional requirements, and fermentation products, five of the eight strains were identified as Butyrivibrio fibrisolvens. Many properties of the remaining three strains resembled Bacteroides ruminicola; however, propionic acid was consistently found as a fermentation product. When the type strains for B. ruminicola subsp. ruminicola and B. ruminicola subsp. brevis were compared with the present isolates, it was found that propionic acid was a normal fermentation product for the type strain B. ruminicola subsp. ruminicola when grown in a 40% rumen fluid-0.5% glucose broth. Production of propionic acid was markedly reduced for all strains when grown in a 20% rumen fluid-1% glucose broth. The three remaining strains were thus placed in the species B. ruminicola, and further classified into the subspecies ruminicola (one strain) and brevis (two strains) on the basis of their requirement for hemin. Although the type strain of B. ruminicola subsp. brevis did not produce propionic acid, both of the present isolates classified as this subspecies produced substantial amounts. One strain of B. ruminicola subsp. brevis had an absolute requirement for volatile fatty acids. Either isobutyric or dl-2-methylbutyric acid would satisfy this requirement, whereas isovaleric acid was ineffective. It is of interest that xylan-fermenting bacteria isolated from 10(-7) and 10(-8) dilutions of rumen contents by use of a xylan medium are similar to the xylan fermenters isolated at the same dilutions with a nonselective medium.     

The use of N,N'-diallylaldardiamides as cross-linkers in xylan derivatives-based hydrogels

N,N′-Diallylaldardiamides (DA) were synthesized from galactaric, xylaric, and arabinaric acids, and used as cross-linkers together with xylan (X) derivatives to create new bio-based hydrogels. Birch pulp extracted xylan was derivatized to different degrees of substitution of 1-allyloxy-2-hydroxy-propyl (A) groups combined with 1-butyloxy-2-hydroxy-propyl (B) and/or hydroxypropyl (HP) groups. The hydrogels were prepared in water solution by UV induced free-radical cross-linking polymerization of derivatized xylan polymers without DA cross-linker (xylan derivative hydrogel) or in the presence of 1 or 5 wt % of DA cross-linker (DA hydrogel). Commercially available cross-linker (+)-N,N′-diallyltartardiamide (DAT) was also used. The degree of substitution (DS) of A, B, and HP groups in xylan derivatives was analyzed according to ¹H NMR spectra. The DS values for the cross-linkable A groups of the derivatized xylans were 0.4 (HPX-A), 0.2 (HPX-BA), and 0.4 (X-BA). The hydrogels were examined with FT-IR and elemental analysis which proved the cross-linking successful. Water absorption of the hydrogels was examined in deionized water. Swelling degrees up to 350% were observed. The swollen morphology of the hydrogels was assessed by scanning electron microscopy (SEM). The presence of cross-linkers in DA hydrogels had only a small impact on the water absorbency when compared to xylan derivative hydrogels but a more uniform pore structure was achieved.     

Thermostable xylanolytic enzymes from Rhodothermus marinus grown on xylan

The thermophilic eubacterium Rhodothermus marinus was cultivated in a fermentor and studied with respect to activities of induced xylanolytic enzymes. Growth in the fermentor on xylan occurred with a maximum specific growth rate of 0.43 h^–1 for a batch culture. The final cell concentration was 4 g cell dry weight (CDW)/l for cells grown on xylan compared to 2 g CDW/l for cells grown without xylan in the cultivation medium. At least two xylanolytic enzymes, endo-1,4--xylanase and xylan 1,4--xylosidase, were secreted into the culture medium when cells were cultivated on xylan. Of the three cellulolytic enzymes tested for activity, -glucosidase activity was in the range of the xylanolytic enzyme activities whereas cellulose-1,4--cellobiosidase and cellulase activities were hardly detectable. The expression of endo-1,4--xylanase activities during cultivation indicates the existance of more than one xylanase in R. marinus. This is also observed in fractions from gel filtration. The xylanolytic enzymes are heat-stable. At 90°C and at pH 7.0 the half-life of the endo-1,4--xylanase was about 14 h and that of xylan 1,4--xylosidase was 45 min. Correspondence to: L. Dahlberg     

X-Ray and stereochemical studies on xylan diacetate

By means of x-ray fiber diffraction, it has been found that xylan diacetate crystallizes with two chains or four residues in a monoclinic cell (space group P2₁): a = 7.64, b = 12.44, c (fiber axis) = 10.31 Å, and γ = 85°. Pairs of residues are related by a twofold screw axis in the c direction. Based on the observed fiber repeat and chain symmetry, the probable conformation of a pair of xylose diacetate residues joined via a β-1,4′ linkage has been obtained by energy minimization methods. The conformations corresponding to a threefold screw axis and a twofold screw axis along the chain have been compared and the reason why xylan diacetate assumes a twofold screw axis seems to be due to intermolecular packing effects rather than intramolecular non-bonded interactions.     

Nanohaloarchaea as beneficiaries of xylan degradation by haloarchaea

Climate change, desertification, salinisation of soils and the changing hydrology of the Earth are creating or modifying microbial habitats at all scales including the oceans, saline groundwaters and brine lakes. In environments that are saline or hypersaline, the biodegradation of recalcitrant plant and animal polysaccharides can be inhibited by salt-induced microbial stress and/or by limitation of the metabolic capabilities of halophilic microbes. We recently demonstrated that the chitinolytic haloarchaeon Halomicrobium can serve as the host for an ectosymbiont, nanohaloarchaeon ‘Candidatus Nanohalobium constans’. Here, we consider whether nanohaloarchaea can benefit from the haloarchaea-mediated degradation of xylan, a major hemicellulose component of wood. Using samples of natural evaporitic brines and anthropogenic solar salterns, we describe genome-inferred trophic relations in two extremely halophilic xylan-degrading three-member consortia. We succeeded in genome assembly and closure for all members of both xylan-degrading cultures and elucidated the respective food chains within these consortia. We provide evidence that ectosymbiontic nanohaloarchaea is an active ecophysiological component of extremely halophilic xylan-degrading communities (although by proxy) in hypersaline environments. In each consortium, nanohaloarchaea occur as ectosymbionts of Haloferax, which in turn act as scavenger of oligosaccharides produced by xylan-hydrolysing Halorhabdus. We further obtained and characterised the nanohaloarchaea–host associations using microscopy, multi-omics and cultivation approaches. The current study also doubled culturable nanohaloarchaeal symbionts and demonstrated that these enigmatic nano-sized archaea can be readily isolated in binary co-cultures using an appropriate enrichment strategy. We discuss the implications of xylan degradation by halophiles in biotechnology and for the United Nation's Sustainable Development Goals.     

A role of xylanase,alpha-L-arabinofuranosidase,and xylosidase in xylan degradation

Renewable natural resources such as xylans are abundant in many agricultural wastes. Penicillium sp. AHT-1 is a strong producer of xylanolytic enzymes. The sequential activities of its xylanase, alpha-L-arabinofuranosidase, and beta-xylosidase on model hemicellulose oat-spelt xylan was investigated. Optimum production of the enzymes was found in culture containing oat-spelt xylan at 30 degrees C and initial pH 7.0 after 6 days. The enzymes were partially purified by ammonium sulphate fractionation and anion-exchange chromatography on DEAE-Toyopearl 650 S. The apparent molecular mass was 21 kDa, and the protein displayed an "endo" mode of action. The xylanase exhibited glycotansferase activity. It synthesized higher oligosaccharides from the initial substrates, and xylotriose was the shortest unit of substrate transglycosylated. Xylanolytic enzymes (enzyme mixture) produced by this Penicillium sp. interacted cooperatively and sequentially in the hydrolysis of oat-spelt xylan in the following order: alpha-L-arabinofuranosidase --> xylanase --> beta-xylosidase. All three enzymes exhibited optimal activity under the same conditions (temperature, pH, cultivation), indicating that they alone are sufficient to completely depolymerize the test xylan. Results indicate that the xylanolytic enzyme mixture of Penicillium sp. AHT-1 could be useful for bioconversion of xylan-rich plant wastes to value-added products.     

Dynamic consolidated bioprocessing for direct production of xylonate and shikimate from xylan by Escherichia coli

Numerous value-added chemicals can be produced using xylan as a feedstock. However, the product yields are limited by low xylan utilization efficiency, as well as by carbon flux competition between biomass production and biosynthesis. Herein, a dynamic consolidated bioprocessing strategy was developed, which coupled xylan utilization and yield optimization modules. Specifically, we achieved the efficient conversion of xylan to valuable chemicals in a fully consolidated manner by optimizing the expression level of xylanases and xylose transporter in the xylan utilization module. Moreover, a cell density-dependent, and Cre-triggered dynamic system that enabled the dynamic decoupling of biosynthesis and biomass production was constructed in the yield optimization module. The final shake flask-scale titers of xylonate, produced through an exogenous pathway, and shikimate, produced through an endogenous pathway, reached 16.85 and 3.2 g L⁻¹, respectively. This study not only provides an efficient microbial platform for the utilization of xylan, but also opens up the possibility for the large-scale production of high value-added chemicals from renewable feedstocks.     

Cellulose and xylan degrading enzymes of Fusarium avenaceum

It was found that crude preparation obtained from the culture medium of Fusarium avenaceum degraded cellulose and xylan. After chromatography on CM-Sepharose CL-6B of this preparation six fraction were obtained. The eluted fractions II and V showed xylanase activity, fraction IV — cellulase activity and fraction III — xylanase and cellulase activity. The end products of xylan hydrolysis by all xylanase fractions (II, III, V) were xylobiose, xylose, xylotriose and xylotetrose. The end products of cellulose hydrolysis by fractions III and IV was cellobiose, glucose and cellotriose. The data from gel filtration on Sephacryl S-200 indicated a molecular weight of more than 250,000 for both cellulase IV and xylanase V. After gel filtration in the presence of urea disaggregation of those high molecular xylanase and cellulase particles was observed. Xylanase II in difference from the other fractions contained higher amount of sugar. Digestion of fraction II with cellulase-hemicellulase preparation from Phoma hibernica decreased the content of sugar from 17% to 8%, but did not change its enzymatic properties. Cellulase IV as well as xylanase V were inactivated by N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and tetranitromethane, hence it is suggested that tryptophan and tyrosine are the essential for the activity of these enzymes.     

Studies on xylan degrading enzyme from Chainia

Summary The sclerotial actinomycete Chainia (NCL 82-5-1) secreted extracellular xylanase in submerged culture in media containing yeast extract and wheat bran or commercial xylan. A high activity (28 IU/ml) of xylanase was obtained in 72 h on a medium containing 3% xylan. Only a single species of xylanase (i.e. without isoenzymes) was detected by polyacrylamide gel electrophoresis. It had an optimum pH of 5.0 and optimum temperature of 65°C. It was stable at pH 6.0 to heating at 60°C for 10 min. Its pI was 8.0 and the K_m was 0.4%. The results are discussed in relation to xylanase reported from actinomycetes such as Streptomyces xylophagus.     

Arabidopsis thaliana IRX10 and two related proteins from psyllium and Physcomitrella patens are xylan xylosyltransferases

The enzymatic mechanism that governs the synthesis of the xylan backbone polymer, a linear chain of xylose residues connected by β‐1,4 glycosidic linkages, has remained elusive. Xylan is a major constituent of many kinds of plant cell walls, and genetic studies have identified multiple genes that affect xylan formation. In this study, we investigate several homologs of one of these previously identified xylan‐related genes, IRX10 from Arabidopsis thaliana, by heterologous expression and in vitro xylan xylosyltransferase assay. We find that an IRX10 homolog from the moss Physcomitrella patens displays robust activity, and we show that the xylosidic linkage formed is a β‐1,4 linkage, establishing this protein as a xylan β‐1,4‐xylosyltransferase. We also find lower but reproducible xylan xylosyltransferase activity with A. thaliana IRX10 and with a homolog from the dicot plant Plantago ovata, showing that xylan xylosyltransferase activity is conserved over large evolutionary distance for these proteins.     

Two Arabidopsis proteins synthesize acetylated xylan in vitro

Xylan is the third most abundant glycopolymer on earth after cellulose and chitin. As a major component of wood, grain and forage, this natural biopolymer has far‐reaching impacts on human life. This highly acetylated cell wall polysaccharide is a vital component of the plant cell wall, which functions as a molecular scaffold, providing plants with mechanical strength and flexibility. Mutations that impair synthesis of the xylan backbone give rise to plants that fail to grow normally because of collapsed xylem cells in the vascular system. Phenotypic analysis of these mutants has implicated many proteins in xylan biosynthesis; however, the enzymes directly responsible for elongation and acetylation of the xylan backbone have not been unambiguously identified. Here we provide direct biochemical evidence that two Arabidopsis thaliana proteins, IRREGULAR XYLEM 10–L (IRX10‐L) and ESKIMO1/TRICOME BIREFRINGENCE 29 (ESK1/TBL29), catalyze these respective processes in vitro. By identifying the elusive xylan synthase and establishing ESK1/TBL29 as the archetypal plant polysaccharide O‐acetyltransferase, we have resolved two long‐standing questions in plant cell wall biochemistry. These findings shed light on integral steps in the molecular pathways used by plants to synthesize a major component of the world's biomass and expand our toolkit for producing glycopolymers with valuable properties.